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| United States Patent |
6,080,573
|
|
Convents
, et al.
|
June 27, 2000
|
Enzymatic oxidation process
Abstract
There is provided an enzymatic oxidation process wherein a substance which
is to be oxidised is reacted with (a) an enzyme exhibiting peroxidase
activity and a source of hydrogen peroxide or an enzyme exhibiting oxidase
activity on phenolic compounds and (b) a compound which enhances the
oxidation activity of the enzyme, characterized in that the compound
specifically binds the substance which is to be oxidized. Furthermore,
there is provided an enzymatic stain bleaching or anti dye-transfer
composition comprising: (a) an enzyme exhibiting peroxidase activity and a
source of hydrogen peroxide or an enzyme exhibiting oxidase activity on
phenolic compounds and (b) a compound which enhances the oxidation
activity of the enzyme and which is capable of binding selectively to a
stain chromophore or textile dye in solution.
| Inventors:
|
Convents; Daniel (Merelbeke, BE);
van Drunen; Rudolf Willem Pieter (Maassluis, NL);
Verrips; Cornelis Theodorus (Vlaardingen, NL)
|
| Assignee:
|
Lever Brothers Company, Division of Conopco, Inc. (New York, NY)
|
| Appl. No.:
|
977586 |
| Filed:
|
November 25, 1997 |
Foreign Application Priority Data
| Current U.S. Class: |
435/263; 435/41; 435/192; 435/264; 510/530 |
| Intern'l Class: |
C11D 003/386; C12P 001/00; C12N 009/08; D06M 016/00 |
| Field of Search: |
435/192,41,264,263
510/530
|
References Cited
U.S. Patent Documents
| 5700769 | Dec., 1997 | Schneider et al. | 435/192.
|
| Foreign Patent Documents |
| 086139 | Aug., 1983 | EP.
| |
| 189687 | Aug., 1986 | EP.
| |
| 266348 | Mar., 1992 | FR.
| |
| 1944904 | Apr., 1971 | DE.
| |
| 282-588 | Dec., 1927 | GB.
| |
| 94/12620 | ., 0000 | WO.
| |
| 91/05839 | ., 0000 | WO.
| |
| 91/05839 | May., 1991 | WO.
| |
| 92/18683 | Oct., 1993 | WO.
| |
| 94/12619 | Jun., 1994 | WO.
| |
| 94/12620 | Jun., 1994 | WO.
| |
| 94/12621 | Jun., 1994 | WO.
| |
| 95/01426 | Jan., 1995 | WO.
| |
| 95/07988 | Mar., 1995 | WO.
| |
| 96/10079 | Apr., 1996 | WO.
| |
| 96/06930 | Jul., 1996 | WO.
| |
| 97/31090 | Aug., 1997 | WO.
| |
Primary Examiner: Lilling; Herbert J.
Claims
What is claimed is:
1. An enzymatic oxidation process wherein a substance which is to be
oxidized is reacted with (a) an enzyme exhibiting peroxidase activity and
a source of hydrogen peroxide or an enzyme exhibiting oxidase activity on
phenolic compounds and (b) a peptide which enhances the oxidation activity
of the enzyme) characterized in that the compound selectively binds the
substance which is to be oxidized and the substance which is to be
oxidized is selected from the group consisting of porphyrin derived
structures, tannins, polyphenols, carotenoids, anthocyanins, maillard
reaction products and textile dyes.
2. Process according to claim 1, wherein the peptide contains one or more
tyrosine residues.
3. Process according to claim 1, wherein the binding compound has a
chemical equilibrium constant K.sub.d for the substance of less than
1*10.sup.31 4.
4. Process according to claim 1, wherein the chemical equilibrium constant
K.sub.d for the substance is less than 1*10.sup.-7.
5. Process according to claim 3, wherein the binding compound has a
chemical equilibrium constant K.sub.d for the substance of less than
1*10.sup.31 6.
Description
TECHNICAL FIELD
The present invention generally relates to an enzymatic oxidation process
wherein a substance which is to be oxidised is reacted with a laccase, or
with a peroxidase and a source of hydrogen peroxide, in the presence of a
compound which enhances the oxidation reaction. More in particular, the
invention relates to an enzymatic detergent composition for stain
bleaching or anti dye-transfer.
BACKGROUND AND PRIOR ART
Peroxidases and laccases are well described as enzymes which can be used to
catalyse the oxidation reaction of a substrate with hydrogen peroxide or
molecular oxygen, respectively. Several applications of these enzymes in
oxidative processes have been described. Such applications include,
amongst others, stain bleaching and anti dye-transfer in detergents,
polymerization of lignin, in-situ depolymerization of lignin in Kraft
pulp, bleaching of denim dyed garments, polymerization of phenolic
substances in juices and beverages and hair bleaching (WO-A-92/18683,
WO-A-95/07988, WO-A-95/01426).
WO-A-91/05839 (Novo Nordisk) discloses enzymatic anti dye-transfer
compositions comprising an (a) an enzyme exhibiting peroxidase activity
and a source of hydrogen peroxide or (b) an enzyme exhibiting oxidase
activity on phenolic compounds. The compositions are said to bleach any
dissolved dye so that no dye can redeposit upon the fabric.
Characteristic to peroxidases and laccases is that they have little
substrate specificity. Most small phenolic molecules are substrates to
these enzymes. The range of molecules which can be oxidized by these
enzymes can be extended by the addition of so-called enhancers. These
molecules are then the primary substrate for the enzymes. Upon reaction
with the enzyme, the enhancers are oxidized to generate radicals which
subsequently oxidize the final substrate of interest.
Several classes of molecules have been described as enhancers for
peroxidases and/or laccases. Among these are simple substituted phenols,
benzidine derivatives, phenothiazine derivatives, and azino compounds
(WO-A-94/12619, WO-A-94/12620 and WO-A-94/12621, all Novo Nordisk). The
value of these enhancers has been demonstrated in anti dye transfer
compositions for detergents.
Whereas enhancers broaden the range of substrates which can be oxidized by
the enzyme, they do not incorporate any substrate specificity in the
oxidation process. To the contrary, addition of enhancers renders the
oxidation reaction more aggressive and difficult to control.
We have now surprisingly found that it is possible to control the enzymatic
oxidation reaction by incorporating substrate selectivity into the
enhancer molecule. The addition of a selective enhancer was found to allow
the tailoring of the otherwise largely random oxidation process.
Moreover, we have identified an experimental procedure which allows the
development of such selective enhancers. We have found that peptides,
which selectively bind the substrate to be oxidized by a peroxidase or a
laccase, can act as such an enhancer. Therefore, for the identification of
selective enhancers, one needs to screen for peptides which bind to the
molecule to be oxidized, and then from those binding peptides, screen
and/or develop a peroxidase/laccase enhancer.
The use of peroxidases and laccases with enhancers has so far most
extensively been described in the areas of pharmaceutical kits and
detergent anti dye-transfer compositions. Especially in the latter
application, incorporation of selectivity in the bleach reaction is of
high value. For dye-transfer prevention, the dye should only be bleached
in solution, without causing dye damage to the fabric. Stain bleaching
compositions should be targeted towards oxidation of the stain
chromophores, as opposed to the dye molecules on the garments.
DEFINITION OF THE INVENTION
According to a first aspect of the invention, there is provided an
enzymatic oxidation process wherein a substance which is to be oxidised is
reacted with (a) an enzyme exhibiting peroxidase activity and a source of
hydrogen peroxide or an enzyme exhibiting oxidase activity on phenolic
compounds and (b) a compound which enhances the oxidation activity of the
enzyme, characterized in that the compound selectively binds the substance
which is to be oxidized.
According to a second aspect, there is provided an enzymatic stain
bleaching or anti dye-transfer composition comprising: (a) an enzyme
exhibiting peroxidase activity and a source of hydrogen peroxide or an
enzyme exhibiting oxidase activity on phenolic compounds and (b) a
compound which is capable of binding selectively to a stain chromophore or
textile dye in solution.
DESCRIPTION OF THE INVENTION
In a first aspect, the invention relates to an enzymatic oxidation process
wherein a substance which is to be oxidised is reacted with (a) an enzyme
exhibiting peroxidase activity an a source of hydrogen peroxide or an
enzyme exhibiting oxidase activity on phenolic compounds and (b) a
compound which enhances the oxidation activity of the enzyme. According to
the invention, the compound which enhances the oxidation reaction is
capable of binding selectively to the substance which is to be oxidised.
The oxidation process can be used within a detergent composition,
specifically suited for stain bleaching and/or dye transfer prevention
purposes, and this constitutes a second aspect of the invention. The
detergent composition may take any suitable physical form, such as a
powder, an aqueous or non aqueous liquid, a paste or a gel.
(a) The enzyme
The enzymatic oxidation composition according to the invention comprises,
as a first constituent, an enzyme. The enzyme may either be an enzyme
exhibiting peroxidase activity (which is then used together with a source
of hydrogen peroxyde), or an enzyme exhibiting oxidase activity on
phenolic compounds, such as phenol oxidase or laccase. Suitable enzymes
are disclosed in EP-A-495 835 (Novo Nordisk). For instance, suitable
peroxidases may be isolated from and are producible by plants or
microorganisms such as bacteria or fungi. Preferred fungi are strains
belonging to the class of the Basidiomycetes, in particular Coprinus, or
to the class of Hyphomycetes, in particular Arthromyces, especially
Arthromyces ramosus. Other preferred sources are Hormographiella sp.,
Myxococcus sp., Corallococcus sp. (WO-A-95/11964), or Soybean peroxidase.
Examples of suitable enzymes exhibiting oxidase activity on phenolic
compounds are catechol oxidase and laccase and bilirubin oxidase. The
laccase can be derived from fungi such as Trametes sp., Collybio sp.,
Fomes sp., Lentinus sp., Pleurotus sp., Rhizoctonia sp., Aspergillus sp.,
Neurospora sp., Podospora sp., Phlebia sp., Coriolus sp., Myceliophthora
sp., Coprinus sp., Panaeolus sp., Psathyrella sp. (WO-A-96/06930).
Bilirubin oxidase can be obtained from Myrothecium sp. or Stachibotrys sp.
The enzymatic oxidation compositions of the invention comprise about 0.001
to 10 milligrams of active enzyme per litre. A detergent composition will
comprise about 0.001% to 1% of active enzyme (w/w). The enzyme activity
can be expressed as ABTS
(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) units. One ABTS
unit represents the amount of enzyme which oxidizes ABTS, resulting in an
increase of 1 optical density at 418 nm in one minute. Conditions for the
activity assay are 2 mM ABTS, 1 mM H.sub.2 O.sub.2, 20 mM Tris, pH 9. The
enzyme activity which is added to the enzymatic oxidation composition will
be about 10 to 10.sup.6 ABTS units per litre, preferably 10.sup.3 to
10.sup.5 ABTS units per litre.
The enzymes used in the present invention can usefully be added to the
detergent composition in any suitable form, i.e. the form of a granular
composition, a liquid or a slurry of the enzyme, or with carrier material
(e.g. as in EP-A-258 068 and the Savinase (TM) and Lipolase (TM) products
of Novo Nordisk). A good way of adding the enzyme to a liquid detergent
product is in the form of a slurry containing 0.5 to 50% by weight of the
enzyme in a ethoxylated alcohol nonionic surfactant, such as described in
EP-A-450 702 (Unilever).
(b) The source of hydrogen peroxide
Another ingredient of the enzymatic anti dye-transfer compositions
according to the invention is a source of hydrogen peroxide. This may be
hydrogen peroxide itself, but more stabilized forms of hydrogen peroxide
such as perborate or percarbonate are preferred. Especially preferred is
sodium percarbonate.
Alternatively, one may employ an enzymatic hydrogen peroxide-generating
system. The enzymatic hydrogen peroxide-generating system may in principle
be chosen from the various enzymatic hydrogen peroxide-generating systems
which have been disclosed in the art. For example, one may use an amine
oxidase and an amine, an amino acid oxidase and an amino acid, cholesterol
oxidase and cholesterol, uric acid oxidase and uric acid or a xanthine
oxidase with xanthine. Preferably, however, the combination of a C.sub.1
-C.sub.4 alkanol oxidase and a C.sub.1 -C.sub.4 alkanol is used, and
especially preferred is the combination of methanol oxidase and ethanol.
The methanol oxidase is preferably isolated from a catalase-negative
Hansenula polymorpha strain. (see for example EP-A-244 920 (Unilever)).
c. The enhancer
The novel oxidation process according to the present invention is based on
the presence of a compound, the peroxidase or oxidase enhancer, which
should be capable of binding selectively to the substance which is to be
oxidised. The enzymatic oxidation composition will comprise about 0.001 to
10 mg per litre.
The degree of binding of a compound A to another molecule B can be
generally expressed by the chemical equilibrium constant K.sub.d resulting
form the following binding reaction:
[A]+[B]=[A::B]
The chemical equilibrium constant K.sub.d is then given by:
##EQU1##
Whether the binding to the substance is specific or not can be judged from
the difference between the binding (K.sub.d value) of the compound to that
substance, versus the binding to the material to which that substance is
applied, or versus other substances one does not want to oxidize. For
substances which occur in stains, the latter material can be envisioned to
be the fabric on which the stain is present, or the dye molecules on
coloured garments. The difference between the two binding constants should
be minimally 100, and preferably more that 1000. Typically, the compound
should bind the coloured substance with a K.sub.d value of 1*10.sup.-4 to
1*10.sup.-6, with a background binding to fabric with a K.sub.d of
1*10.sup.-2 to 1*10.sup.-3. Higher binding affinities (k.sub.d of less
than 1*10.sup.-5) and/or a larger difference between coloured substance
and background binding would increase the selectivity of the oxidation
process. Also, the weight efficiency of the compound in the total
detergent composition would be increased and smaller amounts of the
compound would be required.
Several classes of compounds can be envisaged which deliver the capability
of specific binding to substances one would like to oxidize. In the
following we will give a number of examples of such compounds having such
capabilities, without pretending to be exhaustive.
Antibodies
Antibodies are well known examples of compounds which are capable of
binding specifically to compounds against which they were raised.
Antibodies can be derived from several sources. From mice, monoclonal
antibodies can be obtained which possess very high binding affinities.
From such antibodies, Fab, Fv or scFv fragments, can be prepared which
have retained their binding properties. Such antibodies or fragments can
be produced through recombinant DNA technology by microbial fermentation.
Well known production hosts for antibodies and their fragments are yeast,
moulds or bacteria. A class of antibodies of particular interest is formed
by the Heavy Chain antibodies as found in Camelidae, like the camel or the
llama. The binding domains of these antibodies consist of a single
polypeptide fragment, namely the variable region of the heavy chain
polypeptide (HC-V). In contrast, in the classic antibodies (murine, human,
etc.), the binding domain consist of two polypeptide chains (the variable
regions of the heavy chain (Vh) and the light chain (V1)). Procedures to
obtain heavy chain immunoglobulins from Camelidae, or (functionalized)
fragments thereof, have been described in WO-A-94/04678 (Casterman and
Hamers) and WO-A-94/25591 (Unilever and Free University of Brussels).
Alternatively, binding domains can be obtained from the Vh fragments of
classical antibodies by a procedure termed `camelization`. Hereby the
classical Vh fragment is transformed, by substitution of a number of amino
acids, into a HC-V-like fragment, whereby its binding properties are
retained. This procedure has been described by Riechmann et al. in a
number of publications (J. Mol. Biol. (1996), 259, 5, 957-69; Protein.
Eng. (1996), 9, 6, 531-37, Bio/Technology, (1995) 13, 5, 475-79). Also
HC-V fragments can be produced through recombinant DNA technology in a
number of microbial hosts (bacterial, yeast, mould), as described in
WO-A-94/29457 (Unilever).
Peptides
Peptides usually have lower binding affinities to the substances of
interest than antibodies. Nevertheless, the experiments described in the
examples show that the binding properties of peptides can be sufficient to
deliver the desired selectivity in a oxidation process. A peptide which is
capable of binding selectively to a substance which one would like to
oxidize, can for instance be obtained from a protein which is known to
bind to that specific substance. An example of such a peptide would be a
binding region extracted from an antibody raised against that substance.
Alternatively, peptides which bind to such substance can be obtained by the
use of peptide combinatorial libraries. Such a library may contain up to
10.sup.10 peptides, from which the peptide with the desired binding
properties can be isolated. (R. A. Houghten, Trends in Genetics, Vol 9, no
&, 235-239). Several embodiments have been described for this procedure
(J. Scott et al., Science (1990), Vol. 249, 386-390; Fodor et al., Science
(1991), Vol. 251, 767-773; K. Lam et al., Nature (1991) Vol. 354, 82-84;
R. A. Houghten et al., Nature (1991) Vol. 354, 84-86).
Suitable peptides can be produced by organic synthesis, using for example
the Merrifield procedure (Merrifield, J.Am.Chem.Soc. (1963), 85,
2149-2154). Alternatively, the peptides can be produced by recombinant DNA
technology in microbial hosts (yeast, moulds, bacteria) (K. N. Faber et
al., Appl. Microbiol. Biotechnol. (1996) 45, 72-79).
Pepidomimics
In order to improve the stability and/or binding properties of a peptide,
the molecule can be modified by the incorporation of non-natural amino
acids and/or non-natural chemical linkages between the amino acids. Such
molecules are called peptidomimics (H. U. Saragovi et al. Bio/Technology
(1992), Vol 10, 773-778; S. Chen et al., Proc.Natl.Acad. Sci. USA (1992)
Vol. 89, 5872-5876). The production of such compounds is restricted to
chemical synthesis.
Other Organic Molecules
It can be readily envisaged that other molecular structures, which need not
be related to proteins, peptides or derivatives thereof, can be found
which bind selectively to substances one would like to oxidize with the
desired binding properties. For example, certain polymeric RNA molecules
which have been shown to bind small synthetic dye molecules (A. Ellington
et al., Nature (1990) vol. 346, 818-822). Such binding compounds can be
obtained by the combinatorial approach, as described for peptides (L. B.
McGown et al., Analytical Chemistry, Nov. 1, 1995, 663A-668A).
This approach can also be applied for purely organic compounds which are
not polymeric. Combinatorial procedures for synthesis and selection for
the desired binding properties have been described for such compounds
(Weber et al., Angew.Chem.Int.Ed.Engl. (1995), 34, 2280-2282; G. Lowe,
Chemical Society Reviews (1995) Vol 24, 309-317; L. A. Thompson et al.
Chem. Rev. (1996), Vol. 96, 550-600). Once suitable binding compounds have
been identified, they can be produced on a larger scale by means of
organic synthesis.
Obviously, binding alone of the described compound to a substance one would
like to oxidize will not be sufficient to drive the oxidation process.
Because enzymes like peroxidases and laccases are known to oxidize
substances by a one or two electron oxidation mechanism, the compounds
which add selectivity to the oxidation process should be capable to
transfer one or two electrons from the substance to the enzyme. The
incorporation of electron transfer properties into the binding compound
can be achieved by the addition of amino Acids into peptides which are
known to be important for those properties, e.g. tyrosine, tryptophan,
cysteine, histidine, methionine. For organic compounds, aromatic
structures should be incorporated, preferentially with one or more
heteroatoms (S, N, O).
Several classes of substances one would like to oxidize can be envisaged:
For detergents applications, coloured substances which may occur as stains
on fabrics can be a target. Several types or classes of coloured
substances which may occur in stains can be envisaged, such as indicated
below:
1. Porphyrin Derived Structures
Porphyrin structures, often coordinated to a metal, form one class of
coloured substances which occur in stains. Examples are heme or haematin
in blood stain, chlorophyll as the green substance in plants, e.g. grass
or spinage. Another example of a metal-free substance is bilirubin, a
yellow breakdown product of heme.
2. Tannins, Polyphenols
Tannins are polymerised forms of certain classes of polyphenols. Such
polyphenols are catechins, leuantocyanins, etc. (P. Ribereau-Gayon, Plant
Phenolics, Ed. Oliver & Boyd, Edinburgh, 1972, pp.169-198). These
substances can be conjugated with simple phenols like e.g. gallic acids.
These polyphenolic substances occur in tea stains, wine stains, banana
stains, peach stains, etc. and are notoriously difficult to remove.
3. Carotenoids
(G. E. Bartley et al., The Plant Cell (1995), Vol 7, 1027-1038).
Carotenoids are the coloured substances which occur in tomato (lycopene,
red), mango (.beta.-carotene, orange-yellow). They occur in food stains
(tomato) which are also notoriously difficult to remove, especially on
coloured fabrics, when the use of chemical bleaching agents is not
advised.
4. Anthocyanins
(P. Ribereau-Gayon, Plant Phenolics, Ed. Oliver & Boyd, Edinburgh, 1972,
135-169). These substance are the highly coloured molecules which occur in
many fruits and flowers. Typical examples, relevant for stains, are
berries, but also wine. Anthocyanins have a high diversity in
glycosidation patterns.
5. Maillard Reaction Products
Upon heating of mixtures of carbohydrate molecules in the presence of
protein/peptide structures, a typical yellow/brown coloured substance
arises. These substances occur for example in cooking oil and are
difficult to remove from fabrics.
6. Dyes in Solution
For the prevention of dye transfer from a coloured piece of fabric to other
garments during the wash, it valuable to specifically bleach the dye
molecules in the wash solution. Several types of fabric dyes are used, and
can therefore be envisaged to be a target for the oxidation process: e.g.
sulphur dyes, vat dyes, direct dye, reactive dyes and azoic dyes.
The invention will now be further illustrated in the following,
non-limiting Examples.
EXAMPLE 1
Binding Characteristics of Peptides
The specific binding of peptide #1 (NH2-GGSCGYHYQHCGQG-COOH) to the dye
Reactive Red 6 was measured (the peptide contains one disuphide bridge
through the cysteine residues, sequence of the peptides is given in one
letter amino acid codes). The binding was demonstrated by a specially for
this purpose developed Enzyme Linked Immunosorbent Assay (ELISA).
For the detection of binding, the enzyme Alkaline Phosphatase (AP, 2.5
mg/ml) was conjugated with the reactive dye Reactive Red 6 (RR6, 1.25 mM),
by incubation of the enzyme with the dye during 2 hours, at room
temperature in Borate buffer, 0.1 M, 0.15 M NaCl, pH 8.5. The dye thereby
becomes covalently linked to the amino groups of the enzyme by its
triazine unit. Free dye was separated from the enzyme conjugate by gel
filtration (PD-10 column, Pharmacia). Elisa plates (Polysorb, Nunc) were
coated overnight with 100 .mu.l of a 1 mg/ml peptide solution in Phosphate
buffer, 150 mM NaCl, pH 7.4 (PBS). The peptide coated ELISA plates were
blocked with 2% Bovine Serum Albumin (BSA) in PBS for 1 hour, room
temperature. The Alkaline Phosphatase--RR6 conjugate (AP-RR6) was then
incubated for 1 hour, room temperature, in incubation buffer (0.2 M Tris,
20 mM NaCl, 1% PEG 6000, 5% BSA). The plates were washed plates 3 times
with wash buffer (0.2 M Tris, 60 mM Citrate, 0.1 M NaCl, 0.05% Tween) and
3 times with demineralized water. Bound Alkaline Phosphatase (AP) was then
detected by incubation with the substrate p-nitro-phenyl-phosphate. After
30 minutes, the optical density at 405 nm was measured with a ELISA plate
reader. As a control, Alkaline Phosphatase, not conjugated to the dye, was
used. Furthermore, plates were coated with the peptides Arg-Arg,
Lys-Lys-Lys and Val-Gly-Ser-Glu, to demonstrate the specificity of the dye
binding peptide. The results as optical densities at 405 nm are given in
the table below.
______________________________________
OD 450 nm values
AP-RR6 AP
______________________________________
Peptide #1 2.36 0.047
Arg--Arg 0.09 0.003
Lys--Lys--Lys 0.31 0.023
V--G--S--E 0.03 0.003
______________________________________
EXAMPLE 2
Binding Characteristics of the Peptide
The binding of peptide #1 was further demonstrated by direct measurement of
the binding kinetics of the peptides to the dyes in a IASys Biosensor
(Fisons). By means of the reactive triazine group of the dye, reactive red
6 (RR-6) and reactive red 120 (RR-120) were coupled to an aminosilane
surface cell of the instrument. Dye solutions were 1 mM in 0.1 M borate
buffer, 0.15 NaCl, pH 8.5. The cell was incubated for 2 hours at
37.degree. C. for RR-6 and overnight at 37.degree. C. for RR-120. After
coupling the sample cell was extensively washed with PBS, 0.05% Tween. For
the measurement of the binding affinity between the peptide and the dye,
solutions of increasing concentration of peptide were added to the
cuvette, and binding kinetics were monitored. From these kinetics, the
binding affinities, as equilibrium dissociation constants, were
calculated. The results are shown below. Equilibrium dissociation
constants, K.sub.d, for the reaction
[Peptide]+[Dye]=[Peptide::Dye]
is given by:
##EQU2##
Below are shown the K.sub.d values for the binding of the peptides to the
two different dyes.
______________________________________
K.sub.d values
______________________________________
RR-6 1 .multidot. 10.sup.-4
RR-120
5 .multidot. 10.sup.-5
______________________________________
EXAMPLE 3
Peroxidase Bleach Enhancement by Peptides
Dye bleach experiments were performed using a partially purified peroxidase
derived from an Hormographiella species. The enzyme was purified by
ultrafiltration from the fermentation broth, followed by ion-exchange
chromatography using Q-Sepharose (Pharmacia) at pH 7. Enzyme activity is
expressed as ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)
units. One ABTS unit represents the amount of enzyme which oxidizes ABTS,
resulting in an increase of 1 optical density at 418 nm. Conditions for
the activity assay were 2 mM ABTS, 1 mM H.sub.2 O.sub.2, 20 mM Tris, pH 9.
Bleaching experiments were conducted at 25.degree. C. in 20 mM Phosphate
buffer, set at pH 9. Added peroxidase activity was 60 ABTS units per
millilitre. The peptide GGSCGYHYQHCGQG (one letter amino acid code) was
added as a peroxidase enhancer at a concentration of 100 .mu.M. The
Reactive Black 5 concentration was 30 .mu.M, and the H.sub.2 O.sub.2
concentration was 250 .mu.M.
Bleaching of Reactive Black 5 was monitored by the decrease in optical
density at 590 nm. The enhancing activity of the peptide was compared to
that of the free amino acid tyrosine. As the peptide contains 2 tyrosine
residues, 200 .mu.M of the amino acid was added, as a comparison to 100
.mu.M of peptide. The enhanced bleaching activity at pH 9, 25.degree. C.,
of the peroxidase in the presence of the peptide can be seen from the
table below, which shows the OD reading at 590 nm at the indicate time
intervals.
______________________________________
Enhancer
Minutes after tyrosine
peptide
incubation none 200 .mu.M
100 .mu.M
______________________________________
0 0.651 0.651 0.651
2 0.639 0.606 0.430
4 0.634 0.580 0.344
6 0.631 0.559 0.294
8 0.628 0.540 0.263
10 0.625 0.523 0.241
12 0.623 0.507 0.227
14 0.620 0.494 0.216
______________________________________
EXAMPLE 4
Bleaching of Red Beet Solution with Peroxidase--Peptide Enhanced Reaction
In order to study the selectivity of the peptide enhanced reaction, the
bleaching of a red beet solution with the system was assayed. The extract
of red beet is, as with dyes, susceptible the action of peroxidase
enhancers. The figure below shows that there is not reaction enhancement
of the peptide over tyrosine. Experimental conditions are as in example 3.
______________________________________
Minutes after
Enhancer
incubation none tyrosine
peptide
______________________________________
0 1.09 1.082 1.091
2 1.084 1.076 1.083
4 1.067 1.06 1.063
6 1.033 1.022 1.016
8 0.94 0.906 0.881
10 0.796 0.739 0.709
12 0.663 0.591 0.567
14 0.56 0.488 0.477
______________________________________
EXAMPLE 5
Dye Transfer Prevention
The potential of the enzymatic system to prevent dye transfer was assessed
by washing a coloured swatch in the presence of a white pick-up swatch.
The experiments were performed in 25 ml Phosphate buffer, pH 9, containing
the two swatches of 5.times.5 cm. The experiments were performed using a
partially purified peroxidase derived from an Hormographiella species.
Experiments were performed in the presence of 12 ABTS units/ml. The
fabrics were agitated in the wash solution (25 ml) for 30 minutes at
40.degree. C. The fabrics were line dried and the reflectance spectra were
measured using a Minolta spectrometer. The data thereby obtained was
transferred to the CIELAB L*a*b* colour space parameters. In this colour
space, L* indicates lightness and a* and b* are the chromaticity
coordinates. The colour differences between the control swatch, without
addition the peptide enhancer, and the swatches washed in the presence of
different concentrations of peptide, were expressed as .DELTA.E,
calculated from the following equation:
##EQU3##
The whiteness (.DELTA.L) and the colour difference (.DELTA.E) obtained by
the above method are given in the following Table.
______________________________________
Peptide Tyrosine
Concentration
.DELTA.L
.DELTA.E .DELTA.L
.DELTA.E
______________________________________
25 .mu.M 2.40 2.46 -0.5 0.55
50 .mu.M 2.80 2.85 -0.6 0.63
100 .mu.M 3.50 3.62 -2.0 2.05
______________________________________
The addition of the peptide enhancer results in a clear dye transfer
prevention benefit, resulting in a lighter white swatch. The use of free
tyrosine even results n darkening of the white swatch (negative .DELTA.L).
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